N-deacetylthiocolchicine derivatives, their use and pharmaceutical formulations containing them

ABSTRACT

Disclosed is a series of N-deacetylthiocolchicine derivatives of formula (I) in which:—the linker is a bivalent straight or branched C 1 -C 8  alkyl residue, C 3 -C 8  cycloalkyl, a phenylene or heterocyclic C 4 -C 6  ring;—the G 1  and G 2  junctions, which can be the same or different, are —CO—, —COHN—, —CR 2 — groups in which R 2  is hydrogen or a straight C 1 -C 4  alkyl residue, or the G 1 -linker-G 2  group is the —CO— group. The compounds of formula (I) have antiproliferative, antinflammatory, antiarthritic and antiviral activity.

DISCLOSURE OF THE INVENTION

The present invention relates to N-deacetylthiocolchicine derivatives offormula I

in which:

-   -   the linker is a bivalent straight or branched C₁-C₈ alkyl        residue, C₃-C₈ cycloalkyl, a phenylene or C₄-C₆ heterocyclic        ring;    -   the G₁ and G₂, junctions which can be the same or different, are        —CO—, —CONH—, —CR₂— groups, in which R₂ is hydrogen or a        straight C₁-C₄ alkyl residue,

or the G₁-linker-G₂ group is the —CO— group.

The compounds of formula I have antiproliferative, antiinflammatory,antiarthritic and antiviral activity.

BACKGROUND OF THE INVENTION

Colchicine and thiocolchicine have been known for some time in themedical practice. Colchicine is used in the therapy of gout and relatedinflammatories states. 3-O-Demethylthiocolchicine glucoside is used asmiorelaxant in spasticity and muscle pains due to contractures. However,in both cases the use of these compounds is limited due to their hightoxicity.

Colchicine and thiocolchicine are also known antiblastic compounds, iecompounds that are able to destabilize the microtubules throughinteraction with tubuline. The possible use of a number of colchicine-and thiocolchicine-derivatives as antitumor medicaments has beenstudied. Due to their low therapeutic index none of them has beensuccessful, with the sole exception of demecolchicine, introduced intherapy in the sixties for the treatment of leukemic forms andsubsequently replaced by the more effective Vinca alkaloids.

WO 01/68597 discloses thiocolchicine dimers in which the thiocolchicineresidues are liked through a linear aliphatic amido or amido-ureidobridge.

DISCLOSURE OF THE INVENTION

It has now been found that the compounds of formula I haveantiproliferative activity higher than colchicine and thiocolchicine, inparticular on cells expressing the MDR (Multi-Drug Resistance)phenotype.

The compounds of the invention are also more advantageous than thedimers disclosed in WO 01/68597. Moreover, it has been found that theintroduction of an aromatic basic residue in the linker increases of adimension order the cytotoxicity of the compounds. The introduction oflinkers able to properly orient the thiocolchicine residues in the spaceamplifies the spectrum of activity on resistant tumours. Thecytotoxicity of the compounds of formula I proved comparable to that ofthe most effective antitumour medicaments, whereas the action spectrumis remarkably wider on resistant tumours.

DETAILED DISCLOSURE OF THE INVENTION

Disclosed are N-deacetylthiocolchicine derivatives of formula

in which:

-   -   the linker is a bivalent straight or branched C₁-C₈ alkyl        residue, C₃-C₈ cycloalkyl, a phenylene or a C₄-C₆ heterocyclic        ring;    -   the G₁ and G₂ junctions, which can be the same or different, are        —CO—, —CONH—, —CR₂— groups, wherein R₂ is hydrogen or a straight        C₁-C₄ alkyl residue,

or the G₁-linker-G₂ group is the —CO— group

-   -   with the proviso that, when G₁ and G₂ are both CO, or when G₁ is        —CONH— and G₂ is —CO— the linker is different from an alkyl        residue.

Examples of alkyl bivalent residues comprise straight residues with two,three, four, five or six carbon atoms.

Examples of cycloalkyl groups comprise 1,3-cyclohexylene and1,4-cyclohexylene.

Examples of phenylene groups comprise 1,2-, 1,3- or 1,4-phenylene.

Examples of heterocyclic groups comprise pyridyl, pyrazinyl,pyrimidinyl, piperidinyl, piperazinyl linked to the G₁ and G₂ groupsthrough two carbon atoms of the ring, for example in the positions 3,5or 2,5 or 2,6.

G₁ and G₂ are preferably both CO or CONH.

The linker is preferably a phenylene, cycloalkylene or heterocyclicgroup as defined above, preferably a heterocyclic group comprising atleast one basic nitrogen (pyridyl, pyrimidinyl, pyrazinyl, piperidinyl).

The formulas of some specific compounds of formula I are reportedhereinbelow: TABLE 1

FW = 886 C46H54N4O10S2

FW = 942 C50H62N4O10S2

FW = 914 C48H58N4O10S2

FW = 877 C47H47N3O10S2

FW = 876 C48H48N2O10S2

FW = 878 C48H48N2O10S2

FW = 882 C48H54N2O10S2

FW = 882 C48H54N2O10S2

FW = 682 C48H54N2O10S2

FW = 906 C48H50N4O10S2

FW = 906 C48H50N4O10S2

FW = 863 C47H53N3O10S2

FW = 772 C41H44N2O9S2

FW = 848 C48H52N2O8S2

The compounds of formula I have antiproliferative, antinflammatory,antiarthritic and antiviral activity.

The compounds of the present invention are prepared as described in thefollowing.

The compounds of the invention of formula I in which G₁=G₂=CO(bis-amides) are prepared by reacting N-deacetylthiocolchicine withactivated derivatives of the desired dicarboxylic acids in inertsolvents. Among activated dicarboxylic acids derivatives, particularlypreferred are acid chlorides and mixed anhydrides, particularly withtrifluoroacetic acid. Among inert solvents, particularly preferred arechlorinated solvents. As an alternative, N-deacetylthiocolchicine isreacted with the desired dicarboxylic acid in the presence of DMAP(4-N,N-dimethylaminopyridine) and a condensing agent, DCC(dicyclohexylcarbodiimide), at room temperature, or under heating, andwith vigorous stirring until disappearance of the starting products.

The compounds of the invention of formula I in which G₁=G₂=CONH(bis-ureas) are prepared by reacting N-deacetyl-thiocolchicine with thedesired bis-isocyanate in an inert solvent. When the selected isocyanateis not commercially available, it is generated in situ by Lossenrearrangement of the corresponding hydroxamic acid by treatment with acarbodiimide and a base. Alternatively, the isocyanate is generated bytreatment of the corresponding amide with lead tetraacetate.

The compounds in which G₁ and G₂ are a —CR₂— group (bis-amines) areprepared by reductive amination of N-deacetylthiocolchicine withsuitable dialdehydes. Alternatively, the bis-amines are obtained byalkylation of the thiocolchicine nitrogen with suitable halogen- ortosyl-derivatives. In rare cases the bis-amines are prepared from thecorresponding bis-amides through reduction with boranes.

The compounds with different G₁ and G₂ are prepared in two steps througha combination of the methods cited above.

The activity of the compounds of formula I was evaluated on a largenumber of tumour cells expressing different resistance. The mostinteresting activity has been observed in ovary, colon, liver andpancreas tumour lines.

Table 2 reports IC₅₀ values (expressed in nanomols) of some compounds ofthe invention in comparison with the dimer disclosed in example 3 of WO01/68597.

Table 2—IC₅₀ ¹ (expressed in nMoles) of thiocolchicine dimers after 72hours IC₅₀ (nM) ± S.E. A2780/top A2780/pt (resistant (resistant MCF7MCF7-R A2780 A2780/dx to to Widr Compounds (breast) (MDR+) (Ovarian)(Pgp+) topotecan) tocisplatin) (colon) E.g. 3 of 7.9 ± 0.5   33 ± 2.8238 ± 74 697 ± 192 288 ± 13  252 ± 0.7  351 ± 4  WO 01/68597 E.g. 1 0.7± 0.1   72 ± 4.3   3 ± 0.4 806 ± 286 6.9 ± 4.2 3.7 ± 0.6    25 ± 26.4E.g. 3 1.5 ± 0.1 108 ± 14   8 ± 5.6 922 ± 164 5.5 ± 0.1 23 ± 4.6 48 ± 24E.g. 2  14 ± 0.9 135 ± 11 37 ± 5 1273 ± 382  32 ± 24 26 ± 1.0 230 ± 166E.g. 4 252 ± 38 3841 ± 224  152 ± 36  331 ± 63   650 ± 40 ¹ IC₅₀: concentration that inhibits cell growth by 50% compared with nontreated cells.

Cytotoxic activity was evaluated according to the procedure described byM. C. Alley et al., Cancer Research 1998, 48, 589-601.

All the thiocolchicine dimers with the claimed spacers proved to possessantiproliferative, antiinflammatory and antiviral activity. Thesecompounds are therefore useful in the treatment of neoplasias of variousorigin, deforming rheumatoid arthritis and in the treatment of theKaposi tumour whose retroviral component is ascertained. The inhibitingaction of viral replication combined with the inhibition of cellproliferation in actively proliferating tissues is of particularinterest, considering the origin of several human tumours.

For this purpose, the compounds of the invention will be administered aspharmaceutical compositions suitable for intravenous, parenteral, oral,transdermal administration. A particularly useful aspect of theinvention relates to the preparation of complexes with plasma proteins,in particular engineered human albumin. The protein complexes of thesederivatives are obtained adding to a concentrated solution of albuminthe compound dissolved in dioxane in a time that allows the reactionbetween the involved molecular species. After reacting the two molecularspecies (albumin and the colchicine derivative) under physiological pHand ionic strength conditions, the resulting solution is lyophilized. Ifthe lyophilized solution is prepared under conditions of absolutesterility, it is ready for endovenous injection; the lyophilizedsolution, after dispersion in suitable and compatible excipients, can becompressed and administered through the oral route providing plasmaconcentrations of medicaments close to those obtainable though theparenteral route. As an alternative to the use of plasma proteins, giventhe poor water solubility of the compounds, variously functionalizedcyclodextrins or acryl matrices suitable for parenteral administrationin humans can be advantageously used.

Depending on the administration route, the dosage of the compounds willrange from 1 to 20 mg/m² body area. The preferred administration routesare locoregional injection, intravenous and oral administration.

Examples of compositions comprise freeze-dried vials, supportedlyophilized tablets and drinkable solutions. Any other agents thatdissolve the compounds with to an acceptable extent for administration,such as Tween, Cremophor and suitable mixtures thereof with PEG oralcohols are also comprised in the present invention.

The following examples illustrate the invention in greater detail.

EXAMPLES Example 1 3,5-Pyridinedicarboxylic acidbis-(N-deacetylthiocolchicine) amide

10 g of N-deacetylthiocolchicine are dissolved in 60 ml of methylenechloride. 2.24 g (0.5 eq) of 3,5-pyridinecarboxylic acid, 1.64 g (0.5eq) of N,N-dimethylaminopyridine (DMAP) and 8.3 g (1.5 eq) ofdicyclohexylcarbodiimide (DCC) are then added under vigorous stirring.The mixture is left under stirring, monitoring the reaction by TLC(AcOEt:MeOH 10:1), until disappearance of the reagents (about 12 hours).The solution is then filtered through Celite, washing the pad withmethylene chloride (2×100 ml). The combined organic phases are washedfirst with an equal volume of 1N HCl and then with 1N NaOH. The organicphase is dried over sodium sulfate and evaporated under vacuum. Theresidue is purified by filtration on silica gel (AcOEt:MeOH 10:1). Theresulting product is dried overnight in a static dryer at 40° C. undervacuum to give 7.5 g of final product.

¹HNMR (300 MHz, CDCl₃): 1.00-1.42 (m, 2H), 1.50-2.00 (m, 2H), 2.05-2.30(m, 2H), 2.30-2.50 (m, 2H), 2.52 (s, 3H, SMe), 3.71 (s, 3H, MeO-1), 3.90(s, 3H, MeO-2), 3.96 (s, 3H, MeO-3), 4.80-4.92 (m, 2H, H-7), 6.47 (s,2H, H-8), 7.18 (d, 12.0 Hz, 2H, H-12), 7.38 (d, 12.0 Hz, 2H, H-11), 7.67(s, 2H, H-4), 8.52 (br s, 2H, NH), 9.28 (s, 2H, H-2′+H-6′), 9.60 (s, 1H,H-4′); ¹³CNMR (75 MHz, CDCl₃): 182.64, 164.25, 158.88, 154.12, 152.59,152.34, 151.33, 141.92, 139.33, 135.48, 134.47, 132.74, 129.04, 128.54,127.48, 125.71, 107.74, 61.88, 61.61, 56.49, 53.03, 30.09, 15.41. IR(KBr): 2934, 2854, 1664, 1605, 1540, 1485, 1424, 1403, 1349, 1321, 1266,1235, 1195, 1153, 1137, 1095, 1051, 1021, 978, 921, 842, 796, 703.

Example 2 trans-1,4-Cyclohexanedicarboxylic acidbis-(N-deacetylthiocolchicine) amide

10 g of N-deacetylthiocolchicine are dissolved in 60 ml of methylenechloride. 2.31 g (0.5 eq) of trans-1,4-cyclohexanedicarboxylic acid,1.64 g (0.5 eq) of DMAP and 8.3 g (1.5 eq) of DCC are then added undervigorous stirring. The mixture is left under stirring for 12 hours andmonitored by TLC (AcOEt:MeOH 10:1), then filtered through Celite,washing the pad with methylene chloride (2×20 ml). The combined organicphases are washed with an equal volume of 1N HCl, 1N NaOH and brine,dried over Na₂SO₄. After evaporation of the solvent under reducedpressure, the residue is purified by direct column chromatography(AcOEt:MeOH 12:1). The product is crystallized from methanol (10 v/v)and dried overnight in a static dryer at 40° C. under vacuum to give 6.6g of pure compound.

¹HNMR (300 MHz, CDCl₃): 1.30-1.45 (m), 1.45-1.70 (m, 2H), 1.80-1.89 (m),1.89-2.10 (m), 2.18-2.56 (m), 2.43 (s, 3H, SMe), 3.69 (s, 3H, MeO-1),3.92 (s, 3H, MeO-2), 3.97 (s, 3H, MeO-3), 4.80-4.92 (m, 2H, H-7), 6.47(s, 2H, H-8), 7.09 (d, 12.0 Hz, 2H, H-12), 7.34 (d, 12.0 Hz, 2H, H-11),7.86 (s, 2H, H-4), 8.93 (br s, 2H, NH). ¹³CNMR (75 MHz, CDCl₃): 182.44,176.35, 158.52, 153.80, 152.96, 151.55, 141.97, 139.22, 135.04, 134.69,129.93, 127.09, 126.37, 107.04, 62.39, 61.68, 56.45, 51.13, 44.59,36.92, 30.63, 29.61, 27.84, 15.36. IR (KBr): 3442, 3285, 2934, 2855,1674, 1602, 1532, 1484, 1454, 1424, 1403, 1390, 1348, 1321, 1281, 1256,1236, 1195, 1153, 1136, 1094, 1022, 982, 942, 921, 841, 619, 582.

Example 3 3,5-Benzenedicarboxylic acidbis-(N-deacetylthiocolchicine)amide

The procedure of example 1 is followed starting from 5 g of isophtalicacid to obtain the product as a crystalline solid (yield: 82%).

¹HNMR (300 MHz, CDCl₃): 1.00-1.42 (m, 2H), 1.50-2.00 (m, 2H), 2.05-2.30(m, 2H), 2.30-2.50 (m, 2H), 2.52 (s, 3H, SMe), 3.71 (s, 3H, MeO-1), 3.90(s, 3H, MeO-2), 3.96 (s, 3H, MeO-3), 4.80-4.92 (m, 2H, H-7), 6.47 (s,2H, H-8), 7.18 (d, 12.0 Hz, 2H, H-12), 7.38 (d, 12.0 Hz, 2H, H-11), 7.67(s, 2H, H-4), 8.52 (br s, 2H, NH), 8.05 (m, 12H, H-4′+H-6′), 8.50 (m,1H, H-2′), 7.46 (dd, 8.0 Hz, H-5′).

Example 4 N-Deacetylthiocolchicine 1,4-phenylenediamine bis-urea

2.0 g of N-deacetylthiocolchicine are dissolved in 150 ml of drytetrahydrofuran. 0.5 equivalents of 1,4-phenylene diisocyanate (0.4 g)are then added. The reaction mixture is left under stirring at roomtemperature for two days, monitoring by TLC (DCM-EtOH 95:5, Rf=0.30).The solvent is evaporated off and the crude is recrystallized from ethylacetate to obtain 0.95 g of pure product (38%).

¹HNMR (300 MHz, DMSO-d6): 1.72-1.88 (m), 2.08-2.36 (m), 2.58-2.70 (m),2.42 (s, 3H, SMe), 3.58 (s, 3H, MeO-1), 3.82 (s, 3H, MeO-2), 3.87 (s,3H, MeO-3), 4.24-4.35 (m, 2H, H-7), 6.82 (s, 2H, H-8), 7.17 (d, 12.0 Hz,2H, H-12), 7.27 (d, 12.0 Hz, 2H, H-11), 7.13 (s, 2H, H-4), 8.52 (br s,2H, NH).

Example 5 n-Deacetylthiocolchicine 1,3-phenylenediamine bis-urea

The procedure of example IV is followed starting from 2.0 g ofN-deacetylthiocolchicine and 0.4 g of 1,3-phenylene diisocyanate toobtain the desired product (yield: 42%).

¹HNMR (300 MHz, DMSO-d6): 1.72-1.88 (m), 2.08-2.36 (m), 2.58-2.68 (m),2.43 (s, 3H, SMe), 3.58 (s, 3H, MeO-1), 3.83 (s, 3H, MeO-2), 3.87 (s,3H, MeO-3), 4.24-4.35 (m, 2H, H-7), 6.82 (s, 2H, H-8), 7.18 (d, 12.0 Hz,2H, H-12), 7.28 (d, 12.0 Hz, 2H, H-11), 7.14 (s, 2H, H-4), 8.48 (br s,2H, NH).

Example 6 2,9-Diazasebacic acid bis-(N-deacetylthiocolchicine)amide

The procedure of example IV is followed starting from 2.0 g ofN-deacetylthiocolchicine and 0.4 g of 1,4-butanediisocyanate to obtainthe desired product (yield: 63%, 0.4 g). TLC (DCM-EtOH=95:5) Rf=0.38.

¹HNMR (300 MHz, CDCl₃): 1.80-1.95 (m), 2.38-2.54 (m), 2.70-2.95 (m),3.40-3.60 (m), 2.50 (s, 3H, SMe), 3.69 (s, 3H, MeO-1), 3.95 (s, 3H,MeO-2), 3.98 (s, 3H, MeO-3), 4.54-4.65 (m, 2H, H-7), 6.58 (s, 2H, H-8),7.21 (d, 12.0 Hz, 2H, H-12), 7.43 (d, 12.0 Hz, 2H, H-11), 7.81 (s, 2H,H-4), 8.20 (br s, 2H, NH).

Example 7 1,3-Benzenemethylene-bis-N-deacetylthiocolchicine

2.0 g of N-deacetylthiocolchicine are dissolved in 100 ml of chloroform.0.5 Equivalents of isophtalic aldehyde dimethylacetal and 0.01% ofpyridinium tosylate are added. The mixture is refluxed overnight,allowed to cool down to room temperature and placed in an ice bath. 8Equivalents of sodium triacetoxy borohydride are added and the mixtureis left under stirring for 1 day. The solution is then filtered, washedwith the same volume of 0.1 N HCl and then with saturated aqueous sodiumbicarbonate. The organic phase is dried over sodium sulfate and thesolvent is evaporated off. The crude is purified by flash chromatographyto give 0.49 g of product.

¹HNMR (300 MHz, CDCl₃): 1.00-1.42 (m, 2H), 1.50-2.00 (m, 2H), 2.05-2.30(m, 2H), 2.30-2.50 (m, 2H), 2.52 (s, 3H, SMe), 3.71 (s, 3H, MeO-1), 3.90(s, 3H, MeO-2), 3.96 (s, 3H, MeO-3), 4.80-4.92 (m, 2H, H-7), 6.47 (s,2H, H-8), 7.18 (d, 12.0 Hz, 2H, H-12), 7.38 (d, 12.0 Hz, 2H, H-11), 7.67(s, 2H, H-4), 8.52 (br s, 2H, NH), 7.02 (m, 5H).

Example 8 Injectable Formulation of 3,5-pyridinedicarboxylic acidbis-(N-deacetylthiocolchicine)amide Complexed with Human Albumin

1 g of 3,5-pyridinedicarboxylic acid bis-(N-deacetylthiocolchicine)amide is dissolved in 20 ml of dioxane. The resulting solution is slowlydropped into a 5% physiological solution of albumin to obtain ahomogeneous milky suspension. The mixture is left under stirring insterile conditions for two hours, then lyophilized.

The lyophilized product is ready for administration through injectionroute.

1-9. (canceled)
 10. Compounds of formula I

in which: the linker is C₃-C₈ cycloalkyl, a phenylene or C₄-C₆heterocyclic ring; and the G junctions are both —CO— or —CONH. 11.Compounds as claimed in claim 10 in which the linker is selected from1,3-cyclohexylene and 1,4-cyclohexylene.
 12. Compounds as claimed inclaim 10 in which the linker is selected from 1,2-, 1,3- or1,4-phenylene.
 13. Compounds as claimed in claim 10 in which the linkeris selected from pyridyl, piperidinyl, piperazinyl linked to G₁ and G₂groups in the positions 3,5 or 2,5 or 2,6.
 14. The compounds of formulaI of claim 10 for antitumour, antiarthritis, anti-inflammatory andantiviral use.
 15. Pharmaceutical compositions containing the compoundsof formula I of claim 10 as active ingredients in admixture withsuitable carriers and/or excipients.
 16. Compounds as claimed in claim11 in which the linker is selected from 1,2-, 1,3- or 1,4-phenylene.